DNA methylation by DNA methyltransferases (MTases) is an epigenetic strategy for gene regulation that has been detected in all domains of life. However, detailed studies of MTases have been performed in very few bacterial species, and the targets and cellular roles for a majority of bacterial MTases-an extremely diverse family of enzymes with representatives in almost all sequenced bacteria-remain completely uncharacterized. Furthermore, studies of the roles of MTases genome-wide are rare, and we lack any systematic understanding of the regulatory contributions of these enzymes. In this study, I propose comprehensive analyses of the extent and consequences of DNA methylation in the cholera pathogen, Vibrio cholerae, a genetically tractable bacterial species that encodes a diverse subset of the MTases found in the bacterial kingdom. Until recently, such methylome analyses were not technically feasible; however, the advent of single molecule real time (SMRT) DNA sequencing enables quantitative genome-wide mapping of methylation sites in bacteria. Comparative methylome and transcriptomic analyses, using wt and MTase-deficient V. cholerae grown under a variety of conditions, will be used to define the extent to which methylation varies in response to environmental conditions (a largely unexplored question to date) and the overall impact of such methylation on gene expression. My preliminary analyses have already demonstrated that V. cholerae encodes two MTases, HsdM and VCA0198, that have notable effects upon bacterial gene expression and growth. Planned mechanistic analyses focused on individual regulated genes will define the means by which such methylation governs gene expression, including identification of methyladenine and methylcytosine-sensitive DNA binding proteins. In particular, I plan to i) determine the mechanisms of HsdM-mediated repression of gene expression, ii) interrogate the role of 5mC in the growth and environmental adaptation of V. cholerae, and iii) identify and characterize the effects of novel methylation sensitive DNA-binding proteins on gene expression. Overall, this work will help decipher the epigenetic roles and molecular mechanisms behind methylation mediated gene regulation. This work will likely be extrapolateable to other species, and thus will provide us with a deeper understanding of how DNA methylation functions as a universal mechanism for regulating cellular processes across the bacterial kingdom.